SNP molecular marker located on chromosome 2 and related to scylla paramamosain carapace trailing edge width, detection primer pair, kit and application

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker located on a chromosome 2 and related to the back edge width of scylla paramamosain carapace, a detection primer pair, a kit and application. The SNP molecular marker is characterized in that the 202nd basic group from the 5'end of a nucleotide sequence as shown in SEQ ID NO: 1 is A or T. The SNP molecular marker can predict the carapace trailing edge width character of an individual in the early breeding stage, and is used for molecular marker-assisted selective breeding, improving the breeding efficiency and accuracy of scylla paramamosain, shortening the breeding period, reducing the breeding cost and improving the economic benefit.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biotechnology and molecular markers, and relates to a SNP molecular marker related to the width of the rear edge of the carapace of a mud crab, a detection primer pair, a kit and uses. Background Art

[0002] my country's aquatic industry is undergoing an important stage of pursuing high-quality development, among which the improvement of the seed industry is regarded as a core strategy. With the improvement of social and economic levels, the market demand for the quality of aquatic products is also growing. Scylla paramamosain belongs to the class Crustacea, order Decapoda, family Portunidae, and genus Scylla, and is mainly distributed in Southeast Asia and the southeastern coastal areas of my country. In recent years, the cultivation of Scylla paramamosain has developed rapidly in the southern coastal areas of my country, but the output still cannot meet the growing market demand, the price remains high, and the high-quality crabs on the market are in short supply. As one of the main representatives of marine aquaculture crabs in my country, Scylla paramamosain has rich resources and broad market prospects, and its economic value is significant. The width of the rear edge of the carapace of Scylla paramamosain is regarded as one of the morphological characteristics for judging its size and specifications. The width of the rear edge of the carapace is proportional to the size of Scylla paramamosain, and large-sized Scylla paramamosain is more popular among consumers. Therefore, improving the width of the posterior edge of the carapace of the mud crab is of great significance to improving the economic benefits of the mud crab. It is also an important strategic measure to adapt to market demand and increase product added value.

[0003] However, traditional breeding methods, especially those that rely on artificial selection, face the challenges of high assay costs, time-consuming and labor-intensive, which limits their improvement effects. In order to overcome these challenges and achieve efficient breeding improvements, it is necessary to study the genetic factors affecting the width of the posterior margin of the carapace of blue crabs and explore strategies to improve the trait of the posterior margin width of the carapace using modern breeding techniques. Molecular marker-assisted selection is an advanced genetic improvement technology that can quickly and accurately improve the economic traits of blue crabs. Among them, single nucleotide polymorphism (SNP) markers have become the preferred technology for genetic marker-assisted breeding due to their high richness and automated detection. By identifying SNP loci associated with key economic traits, genetic improvement of these traits can be quickly achieved, providing strong support for the high-quality development of the aquatic industry.

[0004] With the development of high-throughput sequencing technology, whole genome resequencing technology has been widely used in the genetic analysis and genetic improvement of important economic traits of agricultural animals. The identification of the major quantitative trait locus (QTL) and key mutation sites affecting the width of the rear edge of the carapace of mud crabs will greatly accelerate the genetic progress of the width of the rear edge of the carapace of mud crabs, thereby improving the economic benefits of mud crab farming. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the deficiencies and shortcomings of the prior art and provide a SNP molecular marker located on chromosome 2 of the mud crab and associated with the width of the rear edge of the carapace.

[0006] To achieve the above object, the present invention provides a SNP molecular marker associated with the width of the rear edge of the carapace of the mud crab, characterized in that the SNP marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 202nd base from the 5' end is A or T. That is, the 2785785th (2785785bp) nucleotide position on chromosome 2 of the mud crab reference genome (National Center for Bioinformation Database retrieval number: GCF_035594125.1) has an A>T mutation;

[0007] Furthermore, the width of the rear edge of the carapace of individuals with the TT genotype and TA genotype of the SNP molecular marker is significantly greater than that of individuals with the AA genotype.

[0008] Furthermore, the mud crab includes mud crab and its hybrid varieties or strains.

[0009] The present invention also provides a primer pair for detecting the SNP molecular marker, characterized in that the primer pair has a nucleotide sequence shown in SEQ ID NO: 2-3.

[0010] The present invention also provides a kit for detecting the SNP molecular marker, characterized in that it comprises the primer pair.

[0011] The SNP molecular marker, the primer pair or the kit are used in the breeding of mud crabs and their hybrid varieties or strains.

[0012] The present invention also provides a method for detecting the trait of the posterior edge width of the carapace of a pseudo-burrowing mud crab, characterized in that the trait of the posterior edge width of the carapace of the pseudo-burrowing mud crab is determined by detecting the SNP marker on the pseudo-burrowing mud crab to be tested; preferably, the pseudo-burrowing mud crab includes the pseudo-burrowing mud crab and its hybrid varieties or strains.

[0013] Furthermore, the width of the posterior edge of the carapace of the mud crab to be tested is determined by detecting the SNP marker on the mud crab to be tested, including: extracting the genomic DNA of the mud crab to be tested; using the primer pair to perform PCR amplification on the genomic DNA of the mud crab to be tested to obtain a PCR amplification product; sequencing the PCR amplification product to obtain a sequencing result; determining the genotype of the SNP marker of the mud crab to be tested based on the sequencing result; and determining the width of the posterior edge of the carapace of the mud crab to be tested based on the genotype of the SNP molecular marker of the mud crab to be tested.

[0014] Furthermore, the width of the rear edge of the carapace of individuals with the TT genotype and TA genotype of the SNP molecular marker is significantly greater than that of individuals with the AA genotype.

[0015] The present invention also provides a genetic improvement method for mud crabs, characterized in that the method comprises: selecting parent mud crab individuals whose genotypes of the SNP molecular marker are TT and TA genotypes in the parent mud crab population, and eliminating parent mud crab individuals whose genotypes of the SNP molecular marker are AA genotypes, so as to increase the frequency of the allele T at the site generation by generation, thereby improving the carapace posterior edge width trait of offspring mud crabs, and the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 202nd base from the 5' end is A or T.

[0016] The mud crab described in the present invention includes mud crab and its hybrid varieties or strains.

[0017] Based on the whole genome SNP data of mud crab, the present invention successfully obtained a SNP site significantly associated with the width of the rear edge of the carapace by using whole genome association analysis, which can be used for molecular marker-assisted selection to improve the trait of the rear edge of the carapace, and is expected to accelerate the genetic improvement process of the trait of the rear edge of the carapace of mud crab. Through this method, genetic breeding can be carried out more effectively, the economic traits of mud crab can be improved, and the sustainable development of mud crab aquaculture can be promoted.

[0018] The present invention obtains a SNP site significantly associated with the trait of the width of the rear edge of the carapace of the mud crab through whole genome association analysis. The SNP can be used as a genetic marker and applied to marker-assisted selection, thereby accelerating the genetic improvement process of the trait of the width of the rear edge of the carapace of the mud crab. The present invention verifies the relationship between different SNP allele types and the phenotype of the width of the rear edge of the carapace of the mud crab, confirms the effect of the SNP marker on the width of the rear edge of the carapace, and provides a theoretical basis for the breeding application of the SNP marker. Among them, the present invention confirms that the width of the rear edge of the carapace of individuals with TT and AT genotypes is significantly higher than that of individuals with AA genotype. The present invention designs a pair of primers that can be used to detect and identify different allele types of the SNP site. The application of the primer pair provides convenience for SNP typing.

[0019] The present invention provides a variety of breeding methods and application schemes based on the SNP marker, including using the SNP to perform genotyping screening on the population; retaining individuals with excellent genotypes for seed preservation; and performing continuous seed selection and matching aimed at increasing the frequency of the target allele. This provides an operational scheme for breeding the trait of the wide rear edge of the carapace using the SNP marker. The application of the SNP marker can enable the trait of the wide rear edge of the carapace to obtain a controllable genetic improvement direction, which is conducive to the rapid breeding of new varieties (strains) of excellent blue crabs with a wide rear edge of the carapace. This will directly increase the commodity value of a single blue crab, which is of great significance to expanding the breeding scale of the mud crab and increasing economic output. The application of the SNP marker will also promote the development of technologies related to molecular breeding and genetic improvement of the mud crab, and will be conducive to promoting the standardization and large-scale breeding of the mud crab. The SNP marker of the present invention and its breeding application can provide genetic analysis strategies and technical references for molecular breeding of other economic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the Manhattan plot of the genome-wide association analysis on chromosome 2 of the mud crab about the trait of the posterior edge width of the carapace; wherein: the horizontal axis represents the chromosome number of the mud crab; the vertical axis represents the -log10 (P) value. DETAILED DESCRIPTION

[0021] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0022] Example 1: Determination of SNP molecular markers associated with the carapace rear edge width trait

[0023] (1) Experimental animals: 367 mud crabs were selected from a cultured population of mud crabs in Ningde, Fujian. The phenotypic data of the width of the carapace posterior margin of each individual were recorded.

[0024] (2) DNA extraction: Genomic DNA was extracted from the muscle tissue of each individual. The Takara genomic extraction kit was used to extract genomic DNA from the samples of the group. The specific operation method was referred to the instruction manual. The DNA quality and concentration were detected by a spectrophotometer, and the DNA samples were stored at -80°C for future use.

[0025] (3) Whole genome resequencing of mud crab:

[0026] DNA samples were sent to Beijing Novogene Technology Co., Ltd., and the whole genome of the mud crab was sequenced at 9× depth using the paired-end sequencing method on the Illumina platform according to the company's standard process. The sequencing data was aligned by BWA software, and the GATK software was used to mine high-density SNP markers and short fragment insertion / deletion mutations (Indels) in the whole genome, and the number of SNPs obtained was 45,874,945. The SNPs obtained above were quality controlled using plink software, and the number of SNPs obtained after quality control was 2,051,575.

[0027] (4) Whole-genome association analysis: The present invention uses the linear mixed model single-point regression analysis of GCTA software to perform GWAS analysis on the above-mentioned quality-controlled data. The Bonferroni method is used to determine the significance threshold of the degree of association between SNP and the trait of the width of the posterior edge of the carapace. The genome-level significance threshold is 0.05 divided by the number of effective SNP sites (the number of SNPs after filtering by the plink software indep-pairwise50, 10, and 0.2 parameters), that is, the genome-level significance threshold is 5.58e-8, that is, 0.05 / 896055 (the number of effective SNPs); the chromosome-level significance threshold is 1 divided by the number of effective SNP sites, that is, the chromosome-level significance threshold is 1.12e-6, that is, 1 / 896055 (the number of effective SNPs). The GWAS analysis results are as follows Figure 1 As shown, there is a site on chromosome 2 of blue crab that significantly affects the width of the posterior edge of the carapace, and the strongest associated SNP is the A>T mutation at position g.2785785 on chromosome 2.

[0028] (5) Correlation analysis between different genotypes and carapace posterior margin width: According to Table 1 and Figure 1 It can be seen that the molecular marker SNP site g.2785785 A>T is extremely significantly correlated with the carapace posterior width trait (P<0.001), indicating that this molecular marker significantly affects the carapace posterior width trait of the mud crab. Therefore, through auxiliary selection of the mud crab SNP site g.2785785A>T, the carapace posterior width of the population can be gradually improved, thereby accelerating the breeding process.

[0029] In addition, according to Table 1, the width of the rear edge of the carapace of TT and TA types is longer than that of AA type, indicating that TT and TA are beneficial to the width of the rear edge of the carapace. Eliminating the AA genotype of mud crab can bring more economic benefits. In the process of breeding, we need to eliminate the AA type of mud crab for breeding, so as to increase the proportion of TT and TA type individuals in the population generation by generation.

[0030] Table 1 Correlation between the molecular marker SNP mutation site g.2785785A>T and the carapace rear edge width trait

[0031]

[0032]

[0033] Example 2: Amplification and sequencing of target DNA sequences

[0034] (1) Design the primer DNA sequence as follows:

[0035] P001-F: 5'-CGCCACACTTGTCCCTTGTTTT-3' SEQ ID NO: 2;

[0036] P002-R: 5'-ACGCGCACACACACTACCAT-3' SEQ ID NO: 3.

[0037] (2) PCR amplification

[0038] 1 μL DNA template, 10.5 μL double distilled water, 12.5 μL 2×Dream Taq Green PCR Mastermix (Thermofisher), and 0.5 μL primers P001-F and P002-R were added to the 25 μL reaction system. The PCR reaction conditions were: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 s, 60°C annealing for 30 s, 72°C extension for 30 s, 30 cycles; 72°C extension for 10 min.

[0039] (3) DNA sequence determination

[0040] DNA sequencing identification: It was carried out at Novogene Technology Co., Ltd., and the gene fragment was tested for a single positive reaction. The measured sequence was compared with the genome sequence to obtain the mutation of the corresponding SNP site. The sequencing results are as follows:

[0041]

[0042] Note: The W marked in the sequence table is the mutation site, which is displayed in bold font (the mutated base in brackets, i.e., allele mutation), and the underline at the beginning and end of the sequence indicates the position of the designed primer sequence.

[0043] Example 3: Analysis of the effect of molecular marker SNP site g.3076679A>T

[0044] The present invention detects the 202nd base mutation site in the SEQ ID NO: 1 sequence, and preliminarily performs an application of the association analysis between its genotype and the carapace posterior width trait of the mud crab, thereby providing a new molecular marker for the molecular marker-assisted selection of the mud crab. If the present invention selects all AA and TA type individuals of the molecular marker affecting the carapace posterior width trait of the mud crab into TT type individuals, the carapace posterior width of the mud crab can be increased by 3.06 mm and 1.09 mm respectively. Since the carapace posterior width and body weight are in a favorable positive correlation, breeders can increase the body weight by increasing the carapace posterior width of the mud crab. It can be seen that increasing the carapace posterior width of the mud crab has a huge potential to provide benefits to the mud crab breeding industry. Among the SNP molecular marker individuals, by selecting the dominant allele (T) of the SNP of the mud crab, the economic benefits of the mud crab can be finally improved, thereby increasing the benefits of aquaculture.

[0045] Example 4: Farming Group Verification

[0046] Using the above primer pair P001-F and P002-R, 29 mud crabs (six months old) from Fuzhou, Fujian were cultured for molecular markers. The SNP site g.3076679A>T was used for typing. 7 cases were TT, 13 cases were TA, and 9 cases were AA. The width of the rear edge of the carapace of TT and TA type individuals was 8.37mm and 1.25mm wider than that of the rear edge of the carapace of AA type individuals, respectively, and the results were consistent with the actual situation.

[0047] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A SNP molecular marker located on chromosome 2 and associated with the width of the rear edge of the carapace of the mud crab, characterized in that: The SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, wherein the 202nd base from the 5' end is A or T.

2. The SNP marker according to claim 1, characterized in that The width of the rear edge of the carapace of individuals with the TT genotype and TA genotype of the SNP molecular marker is significantly greater than that of individuals with the AA genotype.

3. The SNP molecular marker according to claim 1 or 2, characterized in that: The mud crab includes mud crab and its hybrid varieties or strains.

4. A primer pair for detecting the SNP molecular marker according to any one of claims 1 to 3, characterized in that: The primer pair has the nucleotide sequence shown in SEQ ID NO: 2-3.

5. A kit for detecting the SNP molecular marker according to any one of claims 1 to 3, characterized in that: Comprising the primer pair according to claim 4.

6. Use of the SNP molecular marker according to any one of claims 1 to 3, the primer pair according to claim 4 or the kit according to claim 5 in the breeding of mud crabs and their hybrid varieties or strains.

7. A method for detecting the width of the rear edge of the carapace of a mud crab, characterized in that: By testing the SNP molecular markers of any one of claims 1 to 3 on the mud crab to be tested, the carapace posterior edge width trait of the mud crab is determined; preferably, the mud crab includes the mud crab and its hybrid varieties or strains.

8. The method according to claim 7, characterized in that: include: Extracting genomic DNA of the tested mud crab; Using the primer pair described in claim 4, the extracted genomic DNA is subjected to PCR amplification to obtain a PCR amplification product; Sequencing the PCR amplification product to obtain a sequencing result; Based on the sequencing results, the genotype of the SNP molecular marker of the mud crab to be tested is determined; and based on the genotype of the SNP molecular marker of the mud crab to be tested, the width of the rear edge of the carapace of the mud crab to be tested is determined.

9. The method according to claim 7, characterized in that: The width of the rear edge of the carapace of individuals with the TT genotype and TA genotype of the SNP molecular marker is significantly greater than that of individuals with the AA genotype.

10. A method for genetic improvement of mud crab, characterized in that: The method comprises: selecting parent mud crab individuals whose genotypes of the SNP molecular marker are TT and TA genotypes in the parent mud crab population, and eliminating parent mud crab individuals whose genotypes of the SNP molecular marker are AA genotypes, so as to increase the frequency of the allele T at the site generation by generation, thereby improving the carapace rear edge width-related traits of the offspring mud crabs, wherein the SNP molecular marker is a nucleotide sequence shown in SEQ ID NO: 1, and the 202nd base from the 5' end is A or T.

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